IE42191B1 - 7-methoxycephalosporin compounds - Google Patents

Abstract

1488224 7-Methoxycephalosporins SANKYO CO Ltd 20 Nov 1975 [30 Nov 1974] 47840/75 Heading C2C The invention comprises a process for preparing a 7# - amino - 7α - methoxy - 3 - cephem - 4- carboxylate of general Formula (III) (in which: R1 represents an acetoxy group, a carbamoyloxy group or a 1-methyl-1H-tetrazol - 5 - ylthio group; and Y+ represents a cation), which comprises reacting a 7#-arylmethyleneamino - 7α - methoxy - 3 - cephem - 4- carboxylate of general Formula (II) (in which B1 and Y+ are as defined above; n is 1 or 2; and Ar represents a substituted or unsubstituted, monocyclic or fused polycyclic benzenoid ring system) with a hydrazine compound. The invention also comprises a process for preparing compounds (I) (in which R represents cyanomethylthio, 1- cyanoethylthio, azidomethylthio, propargylthio, 2 - hydroxyethylthio, 2,3 - dihydroxypropylthio, methylsulphonyl, ethylsulphonyl, cyanoethylsulphonyl, isoxazol - 3 - yloxy, isoxazol - 3 - ylthio, 1,3,4 - thiadiazol - 2- ylthio, imidazol - 2 - ylthio or sydnon - 3 - yl) which comprises reacting (III) with a carboxylic acid of general Formula (IV) (in which R6 represents the group R or the group R in which any hydroxy groups have been protected) or with a functional equivalent of said acid (IV) to produce a compound of general Formula (V) (in which R1 and R6 are as defined above) and, if necessary, removing any protecting groups in the group R6. Compounds (II) are made by treating corresponding 7 - arylmethyleneamino - 3 - cephem - 4- carboxylates (obtained by Schiff base formation) with oxidizing agent and methanol.

Description

The present invention relates to certain 7j3-amino7a-methoxy-3-cephcm-4-carboxylates having the group -+ + COO Y (in which Y represents a cation) in the 4-position, to a process for preparing these compounds and to the use of these compounds to prepare valuable.cephalosporin antibiotics having a 7a-methoxy group, o Many cephalosporin derivatives are valuable antibiotics and it has recently been discovered that cephalosporin derivatives having an alkoxy group, particularly a methoxy group, in the 7a- position have particularly valuable antibiotic properties. A number of different processes has been proposed for introducing an alkoxy group into the 7aposition of the cephem nucleus of various cephalosporin derivatives; such processes include the following: I5 (a) Diazotization of 7-aminocephalosporanic acid and subsequent conversion of the product into the corresponding alkoxy derivative, as is described in Japanese Patent Provisional Publication No. 931/72 and J. Amer. Chem. Soc., 94, 1408 (1972). (b) Alkylthionation or fluorination and acylation of a 7-benzylideneamino compound and conversion of the product into the corresponding alkoxy derivative, as described in J. Org. Chem., 38, 943 and 2857 (1973), (c) Reaction of a 7-benzylideneamino compound with a dialkyl peroxy compound, as described in Japanese Patent Provisional Publication No. 42691/72. (d) N-Chlorination of a 7-acylaminocephem compound, subsequent conversion of the product to the corresponding acylimino compound, followed by addition of methanol, as described in J. Amer. Chem. Soc., 95, 2401 and 2403 (1973).

However, these known processes have a number of disadvantages, including the need for many reaction stages and complicated procedures, with relatively poor yields and large quantities of by-products.

We have now discovered a relatively simple method of producing cephalosporin antibiotics having a methoxy group in the Ta- position, which can be carried out easily and which produces relatively few by-products compared with prior methods.

Thus, the present invention consists in a process for preparing a 73-amino-7o-methoxy-3-cephem-4-carboxylate of general formula (III): (in which: R* represents an acetoxy group, a carbamoyloxy group or a l-methyl-lH-tetrazol-5-ylthio group; · and Y+ represents a cation) which comprises reacting, a 7β-arylmethy1eneaminff - 7a-methoxy-3-cephem-4-carboxylate of general formula (II): (in which: R^ and Y+ are as defined above; n is 1 or 2; and Ar represents a substituted or unsubstituted, monocyclic or fused polycyclic benzenoid ring system) with a hydrazine compound. 3191 The 7/3-amino-7a-methoxy-3-cephem-4-carboxylate (HI) so prepared may then be used to prepare a 7a-methoxycephalosporin of general formula (I): OCH3 g RCH2C0HH“r—-ζ \ • COOH (I) (in which: R represents a cyanomethylthio group, a 1-cyanoethylthio group, an azidomethylthio group, a propargylthio group, a 2-hydroxyethylthio group, a 2, 3-dihydroxypropylthio group, a methylsulphonyl group, an ethylsulphonyl group, a cyanoethylsulphonyl group, an isoxazol-3-yloxy group, an isoxazol-3-ylthio group, a 1, 3,4-thiadiazol~2-ylthio group, an imidazol-2-ylthio group or a sydnon-3-yl group; and r1 is as defined above) or a salt thereof by the following procedure: (1) reacting the 7/?-amino-7a-methoxy-3-cephem-4carboxylate (III) with a carboxylic acid of general formula (IV): R6CH„COOII (IV) u β (in which R represents the group R or the group R in 4219 1 which any hydroxy groups have been protected) or with a functional equivalent of said acid (II7) to produce a compound of general formula (V); OCH3 R6CH2C0HfJ- ! ' fVl CQOH (in which R^ and R^ are as defined above); (2) if necessary, removing any protecting groups θ from the group R ; and (3) optionally, before or after step (2), reacting said compound (I) or (V) with a base to produce a salt thereof.

The integer nisi or 2 and, when n is 2, the group (Λϊ-)^ may represent a biphenyl, binaphlbalene or p-(4-naphtbyl)benzone system. 4 We particularly prefer that R and R should be sterically hindered alkyl groups, such as isopropyl or t-butyl groups, in which case R and R are most preferably both hydrogen atoms.

The 78-arylmethyleneamino -7a-methoxy-3-cephem-4carboxylates of general formula (II) may be prepared by oxidizing a corresponding 7- arylmethyleneamino-3-cephem-4cnrbuxylate and reacting the resulting product wil.li methanol, as described in co-pending British Patent Specification No. 46592/75.

+ Y in formulae (II) and (III) represents a cation, preferably an alkali metal ion or ammonium ion having the formula: Rb Rd in wiiich Ra, r\ Rc and R^ are the same or different 43191 In the starting material for the process of the invention, the 7/3-aryl methyl eneamino-7ff-methoxy-3cephem-4-carhoxylate (II), the group Ar preferably represents a group of formula: 3 4 5 in which R , R , R and R are the same or different and each represents a hydrogen atom, an alkyl group having from 1 to 4 carbon atoms, which may be a straight or branched chain alkyl group, such as methyl, ethyl, propyl, isopropyl or t-butyl,· an alkoxy group having from 1 to 4 carbon atoms, which may be a straight or branched chain alkoxy group, such as methoxy, ethoxy, propoxy, isopropoxy or t-butoxy; a halogen atom, such as chlorine or bromine; a cyano group; or an alkoxycarbonyl group having from 1 to 4 carbon atoms in the alkyl moiety, such as methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl; or R2 and R3 together and/or R4 and R5 together may form, together with the carbon atoms to which they are joined, a carbocyclic or heterocyclic saturated, unsaturated or aromatic ring, such as a cyclopentane, cyclohexane, cycloheptane or benzene ring. and each represents: a hydrogen atom; a straight or branched chain alkyl group having from 1 to 8 carbon atoms, such as a methyl, ethyl, propyl, isopropyl, t-butyl, octyl or t-octyl group; a cycloalkyl group having from to 7 carbon atoms, such as a cyclopentyl, cyclohexyl or cycloheptyl group; a phenyl group; or a phenylalkyl group having from 1 to 4 carbon atoms in the alkyl moiety, a. b such as a benzyl or phenethyl group; or R and R may form, together with the nitrogen atom to which they are attached, a heterocyclic ring, preferably a saturated heterocyclic ring, such as a pyrrolidine, piperidine or morpholine ring; ρΐ R , R° and Rc may form, together with the nitrogen atom to which they are attached, a heterocyclic ring, preferably a heteroaromatic ring, such as a pyridine, picoline, lutidine, quinoline, quinaldine or + pyrimidine ring. Most preferably, Y represents a lithium, sodium, t-butylammonium, t-octylammonium, dicyclohexylammcnium, diisopropylammonium, triethylammonium or trirnethylbenzylammonium cation.

To prepare the 7/3-amino-7n-methoxy- 3-cephem-4carboxylate (III), a 73-aryllnethyleneann‘no-!7ff-methoxy-3cephem-4-carboxylate (II) is contacted with a hydrazine compound, preferably in the presence of an inert organic solvent. There is no particular limitation on the naturei of the solvent, provided that it does not take part in the reaction. Examples of suitable solvents include alkanols having from 1-4 carbon atoms, such as methanol, ethanol propanol, isopropanol, butanol, isobutanol and pentanol; aromatic hydrocarbons, such as benzene, toluene and xylene; halogenated hydrocarbons, such as chloroform and methylene chloride; dimethylformamide; dimethylacetamide; or mixtures of any two or more thereof.

The hydrazine compound used in this process may be substituted or unsubstituted; examples include phenylacetic acid hydrazide, hydrazine itself, hydrazine hydrate, phenylhydrazine, 2,4-dinitrophenylhydrazine, carboxymethyl pyridinium chloride hydrazide (Girard reagent P), or carboxymethyltrimethylammonium chloride hydrazide (Girard reagent T); The reaction temperature is not particularly critical to the process and the reaction is normally therefore carried out at about ambient temperature, although temperatures both above and below ambient may also be employed.

The time required for the reaction will depend mainly upon the natures of the 7/3-arylmethyleneamino-7ff-inethoxy-3-cephem10 4-carboxylnl<> (II), tlie hydrazine compound and the solvent arid upon the reaction temperature, but the reaction will normally require several tens of minutes. When the reaction is complete, the product may be recovered by conventional means; the reaction mixture may, however, be employed directly for preparing the 7oi-methoxycephalosporin (I) without intermediate isolation of the 7/3-amino-7»-methoxy-3-cephein-4-carboxylate (III), The 7(i>-inethoxyccphalosporin (i) is prepared by reacting a 7/5-amino~7ci-methoxy-3-cephem-4-carboxylate (ΠΙ), which may be prepared as described above, with a carboxylic acid (IV) or with a functional derivative thereof, suitably in an appropriate solvent.

Examples of carboxylic acids having the formula (IV) are: cyanomethylthioacetlc acid, 1-cyanoethylthioacetic acid, azidomethylthioacetic acid, propargylthioacetic acid, methylsulphonylacetic acid, ethylsulphonylacetic acid, cyanoethylsulphonyl acetic acid, isoxazol-3-yloxyacetic acid, isoxazol-3-ylthioacetic acid, 1, 3, 4-thiadiazol-2-ylthioacetic acid, imidazoi-2ylthioacetic acid and sydnon-3-ylaeetic acid. In place of the carboxylic acid of formula (IV) a functional derivative of the acid may be employed. In relation to the carboxylic acitl (IV), the term functional derivative is defined to mean any compound which, under the conditions of the reaction with the 7£-amino-7a-methoxy-3-eephem4-carboxylate (III), will react as, or will react to form, the carboxylic acid of formula (TV). Examples of such functional derivatives thus include: the acid halides of the carboxylic acids (IV), e. g. the acid chlorioe, acid bromide or acid fluoride; the azides of carboxylic acids (IV); mixed acid anhydrides of the acid (IV) with a fatty acid or with an alkyl chlorocarbonate; the simple anhydrides of the carboxylic acids (IV); and reactive esters of acid (IV), e. g. the p-nitrophenyl ester, the cyanomethyl ester or the phthalimide ester.

In formula (IV), the group R may be the same as the group R desired in the final product. However, if the group R contains hydroxy groups, which might undergo reaction under the conditions of the process, these groups are preferably protected by a suitable protecting group; thus, if 2-hydroxyethylthioacetic acid or 2, 3-dihydroxy20 propylthioacetic acid or a functional derivative thereof is to be used in the process, the hydroxy groups are preferably first protected with a suitable protecting group, such as a tetrahydropyranyl group.

The reaction is preferably effected in the presence of a solvent, although the nature of the solvent is not critical, provided that it does not participate in the reaction; examples of suitable solvents include: halogenated hydro5 carbons, such as chloroform, methylene chloride and ethylene chloride; dialkyl ketones having 1-4 carbon atoms in each alkyl moiety, such as acetone or methyl ethj'l ketone; acetic acid esters, such as ethyl acetate or butyl acetate; ethers, such as diethyl ether, θ tetrahydrofuran or dioxan; aromatic hydrocarbons, such as benzene or toluene; acetonitrile; dimethylformamide; dimethylacetamide; water; or a mixture of any two or more thereof. The solvent most suitable for the reaction will depend upon the nature of the ] 5 carboxylic acid or functional derivative thereof employed.

The reaction temperature is not particularly ci'itical and we normally prefer that the reaction should be effected at a temperature between 0°C and ambient temperature, although other temperatures may also be employed. The time required for the reaction will depend upon the nature of the carboxylic acid (IV) or functional derivative thereof, the nature of the 7/3-amino-7n'-methoxy-3-cepliem-4~earboxylatc (III), the solvent and the reaction temperature, but the reaction will normally take from several minutes to several tens of minutes.

If the free carboxylic acid of formula (IV) is used in the reaction, the reaction is preferably carried out in the presence of a condensing agent, for example: a carbodiimide, such as N, N'-dicyclohexylcarbodiirnide; a carbonyldiimide, such as carbonyldiimidazole or carbonylditriazole; or an alkoxyacetylehe, such as methoxyacetylene. If, on the other hand, the acid halide, which is the preferred functional derivative of carboxylic acid (IV), is used, the reaction is preferably carried out in the presence of an acid binding agent, such as an inorganic or organic base. Examples of inorganic bases are alkali metal or alkaline earth metal compounds, for example: the hydroxides, such as sodium hydroxide, lithium hydroxide or potassium hydroxide; the carbonates, such as sodium carbonate or potassium carbonate; or the hydrogen carbonates, such as sodium hydrogen carbonate or lithium hydrogen carbonate. Examples of organic bases are the tertiary amines, such as triethylamine, tributylamine, diethylaniline, N-methylpiperidine, N-methylmorpholinc; pyridine; or quinoline, Iliethylaniline is most preferred.

After completion of the reaction, the 7¢-methoxycephalosporin (I) may be recovered from the reaction mixture by conventional methods; for example, when the reaction is complete, an alkanol having 1-4 carbon atoms (such as methanol) or dilute hydrochloric acid is added to the reaction mixture and the organic layer is extracted with an aqueous solution of disodium hydrogen phosphate, the extract is acidified (e.g. to pll 2) by an appropriate acid and re-extracted with, for example, ethyl acetate, the organic solvent layer is separated and dried, and the solvent is removed, giving the desired product. Conventional purification techniques, such as recrystallization or column chromatography, may then be applied in order to obtain a pure substance.

Examples of 7w- methoxy cephalosporin compounds which may be produced by the process of the invention are as follows: 1. 3~Acetoxymethyl-7/3-azidomethylthioacetamido-7«methoxy-3-cephem-4-carboxylic acid. 2. 3-Acetoxymethyl-7/l-cyarioethylsulphonylacetamido-7«-inethoxy3-cephem-4-carboxylic acid, 3. 3-Acetoxymethyl-7/j-c.yariomethylthioacetamido-7 4. 3-Acetoxymethyl-7/3-(2, 3-dihydroxypropyl Jthioacetnmido7»-methoxy- 3-cephem-4-carboxylic acid. . 3-Acetoxymethyl-7/j-imidazol-2-ylthioacetamido-7nmethoxy-3-cephem-4-earboxylic acid. 6. 3-Acetoxymethyl-7,8-iSoxazol-3-yloxyacetamido-7o'methoxy-3-cephem-4-carboxylic acid. 7. 3-Aeetoxymethyl-7/3-isoxazol-3-ylthioaeetamido-7o:methoxy-3-cephem-4-carboxylic acid. 8. 3-Acetoxymethyl-7c'-methoxy-7/3-propargylthioacetamido- 3-cephem-4-carboxylic acid. 9. 3-Acetoxymethyl-7a-methoxy-7^-(l, 3,4-thiadiazol-2-ylthioacetamido) - 3-cephem-4-carboxylic acid. . 7/3-Azidomethylthioacetamido-3-carbamoyloxymethyl10 7ff-methoxy-3-cephem-4-carboxylic acid. 11. 3-Carbamoyloxymethyl-7/3-cyanoethylsulphonylacetamido7o'-methoxy-3-cephem-4-carboxylic acid. 12. 3-Carbamoyloxymethyl-7/3-cyanomethylthioacetamido7c-methoxy-3-cephem-4-carboxylic acid. 13. 7/3-Azidomethylthioacetamido-7a- methoxy- 3-(1 -methyllH-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid, 14. 7/3-Cyanoethylsulphonylacetamido-7a-methoxy-3(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid. . 7jl-(l-Cyanoethylthioacetamido)-7fi'-methoxy-3(1-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem*4-carboxylic acid. 16. 7/3- Cyanomethylthioacetamido- 7 a- methoxy- 3-(1- methyllH-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid. 17. 7/?-3£thylsulplionylaeetamido-7Q:-niiithoxy-3-(l-methyllII-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid. 18. 7/1- (2-Ilydroxyeihyl)thioacetamido- 7a-methoxy- 3(1 - methyl- ΙΗ-tctrazol - 5-ylthiomethyl)- 3-cephem-4-carboxylic acid. 19. 7p-Imidazol-2-yltliioacetamido-7a-methoxy-3(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid. . 7/3-Isoxazol-3-yloxyacetaniido~7»'-inethoxy-3-(lmethyl-lII-tetrazol-5-ylthiometliy!)~3-cephem-4-carboxylic acid, 21. 7/3-Isoxazol-3-ylthioacetamido-7a-methoxy-3-(lmethyl-ΙΠ-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid. 22. 7o'-Methoxy-73-rnethylsulphonylacetamido-3-(lmethyl-12I-tctrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid. 23. 7ff-Methoxy-3-(l-methyl-in-tetrasol-5-ylthiotnethyl)7/l-propargyllhioacetamido-3-cephem-4-carboxylic acid. 24. 7tf-Methoxy-3-(l- methyl-ljl-tetrazol-5-yltbiometbyl )7/>-sydnon-3-ylacetaniido-3-cepheni~4-carboxylic acid, . 7a-Methoxy- 3-(1 -methyl-111-tetrazol-5-ylthiomethyl)7/1-(1,3,4-thiadjazol-2-ylthioacetamido)-3-cephem-4carboxylic acid.

These compounds are hereinafter referred to by the numbers given above. Of these compounds, the following have excellent antibacterial activity and are particularly preferred: Nos. 3, 7, 10, 12, 13, 14, , 16, 17, 18, 20, 21, 22, 23, and 24. The 7a-methoxycephalosporins of general formula (X) have a broad antibacterial spectrum and have particularly good antibacterial activity against both Gram-positive and Gram-negative organisms compared with other 7a-methoxy10 cephalosporin derivatives. The salts of these compounds also share their good antibacterial activity.

The minimum inhibitory concentrations (meg/ml) were determined for certain of the Ta-methoxycephalosporins of general formula (I) and the results are shown in the following Table. These results show that the compounds have excellent antibacterial activity against a wide range of pathogenic microorganisms. In the Table, the microorganisms are identified by the following codes: IA = Staphylococcus aureus 209 P IB = Staphylococcus aureus (CP and PC resistant) HA = Escherichia coli NIHJ ΙΠ3 = Escherichia coli 609 (CER resistant) III = Shigella flexneri Komagome IVA = Klebsiella pneumoniae 806 IVB V = VI Klebsiella pneumoniae 84G {CER resistant) Proteus vulgaris Salmonella enteritidis Gartner TABLE Compound No. Microorganism IA IB ΠΛ IIB III IVA IVB V VI 1. 0.4 0.8 6.2 6.2 6.2 6.2 >200 3.1 1.5 3. 0.2 0.8 1.5 1.5 3.1 1.5 >400 6.2 0.8 7. 0.2 0.8 3.1 6.2 6.2 3.1 >400 3.1 1.5 8. 1.5 6.2 6.2 12.5 12.5 12.5 >200 12.5 6.2 10. 0.2 1.5 3.1 6.2 3.1 3.1 >400 3.1 1.5 12. 0.8 3.1 1.5 1.5 3.1 3.1 . >400 6.2 1.5 13. 0.4 0.4 3.1 3,1 1.5 3.1 >200 1.5 ! 0.8 1 1 14. 1.5 3.1 1.5 1.5 3.1 1.5 200 6.2 1 0.8 15. 0.4 0.8 1.5 3.1 1.5 1.5 400 0.8 0.4 10. 0.2 0.8 0.8 0.8 0.8 0.8 >200 1.5 .0.2 17. 1.5 1,5 1.5 1.5 1.5 1.5 >400 6.2 0.8 18. 0.8 1.5 1.5 1.5 1.5 1.5 200 3.1 0.8 20. 0.2 0.8 3.1 3.1 3.1 3.1 >400 6,2 1.5 21. 0.2 0.8 3.1 3.1 3.1 6.2 >400 3.1 0.8 22. 0.8 3.1 1.5 1.5 1.5 0,8 400 12.5 0.4 23. 0,2 0.8 3.1 3.1 3.1 3.1 400 3.1 0.8 24. 0.8 1.5 1.5 1.5 1. 5 1.5 >200 6,2 0.8 43191 For the treatment of infection, the 7tt-methoxycephalosporin compounds (I) are preferably administered orally or parenterally, as is conventional for the administration of antibiotics; preferred formulations are tablets, capsules, injectible liquids and suspensions, tablets or injectible liquids normally being preferred. The compounds are most preferably administered by injection. The daily dosage of the 7a'~methoxycephalosporin compounds (I) will, naturally, vary depending upon the age, condition and body weight of the patient. However, the compounds will normally be administered in divided doses of from 250 mg to 3000 mg per day for adults, normally three or four times a day, although, if necessary, amounts above 3000 mg per day may be administered.

The processes of the present invention are further illustrated with reference to the following Examples; the preparation of the 7/3-arylmethyleneamino-7o'-methoxy-3cephem-4-carboxylate (II) starting material sis illustrated in the following Preparations.

PREPARATION 1 Dicyclohexylammonium 3-acetoxymethyl-7,'?-(4-hydroxy-3, 5di-t-butylbenzylideneamino)-7g-methoxy-3-cephem-4carboxylate 29, 2 g of 3-acetoxymethyl-7^-amino-3-cephem-4carboxylic acid were suspended In 600 ml of methanol, and 18,1 g of dicyclohexylamine were added to the resulting suspension. The mixture was then stirred for 1 hour at room temperature to obtain a semi-transparent solution, to which were then added 24. 0 g of 4-hydroxy-3, 5-di-tbutylbenzaldohyde; the mixture was stirred for a further hours at room temperature, 50 g of molecular sieve 3A were added and stirring was continued for a further 5 hours. At the end of this time, the reaction mixture was filtered and the solvent was evaporated under reduced pressure from the filtrate to give a crystalline substance which, after addition of isopropanol, was collected by filtration, yielding 45. 03 g of dicyclohexylammonium 3-acetoxym:cthyl-7/3-(4-hydroxy-3, 5-di-t-butylbenzylidene20 amino)-3-cephem-4-carboxylate. 13.4 g of this dicyclohexylammonium 3-acetoxymethyl-7/3-(4-hydroxy3, 5-di-t-butylbenzylideneamino)-3-cephem-4-carboxylate were dissolved in 200 ml of methanol and the resulting solution was cooled to -15°C. To this solution was added dropwise', over about 5 minutes, a solution of 4,74 g of 2, 3(iicliloro-5, G-dicyano-1,4-benzoquinonc in 40 ml of methanol. Stirring was continued at the same temperature for a further 10 minutes, aftex· which 1 g of 2, C-di-t-butyl-£-cresol was added to the reaction mixture. After stirring for a further 10 minutes at 0°C, 5, 60 ml of triethylamine were added to the reaction mixture, which was then condensed to about 50 ml by evaporation under reduced pressure. 300 - 400 ml of chloroform were added to the mixture, which was then washed three times with the same volume of water and dried over anhydrous magnesium sulphate, The solvent was then removed from the dried mixture by evaporation under reduced pressure, leaving a residue which was extracted with diethyl ether. The ethereal extract was condensed and n-hexane was added, giving a precipitate which was collected by filtration to yield 13, 03 g of dicyclohcxylainrnoniuni 3~acetoxyinelhy]-7/i-(4-hydroxy-3, 5-di-tbutylbenzylideneamino)-7a'-methoxy-3-eopiicm-4-carboxylate as a yellow powder. A pure rumple of this salt having a melting point of 139 - 141 °C was obtained by recrystallization from a mixture of chloroform and n-hexane.

UV spectrum (Till·') λ : 2ii3 rim.

(TIIF - tetraliydrofu an) NMR spectrum (CDC1 ) 6 ppm: ) V 8.38 , (singlet, -CH=N- ),· 7.57 l (singlet, benzene ring H); 5.52 (broad, OH); 5 4. 96 (singlet, H at 6- position),- 4.87 (singlet, -CH - at 3- position); 3.47 (singlet, OCHg ); 3.23 (double doublet, Hg at 2- position); 1.92 (singlet, CHgCO); 10 1.40 (singlet. t-butyl).

Thin layer chromatography (silica gel) developing solvent chloroform/methanol (4 : 1 by volume): Rf : 0.74.

Elemental analysis: Calculated for Cgg Hg? Ng O^S. 1/2 : C, 66.29%; H, 8.68%; N, 5.66%; S, 4.31%.

Found: C, 66.12%; H,8.64%; N,5.70%; S. 4. 20%.

PREPARATION 2 Lithium 3-acetoxymethyl-7/3-(4-hydroxy-3, 5-diisopropyl20 benzylideneamino) - 7 a- methoxy- 3 - cephem- 4- carboxylate 544 mg of 3-acetoxymethyl-70-amino-3-cephem-4carboxylic acid were suspended in 8 ml of methanol; to this suspension were added dropwise a solution of 14 mg of lithium in 2 ml of methanol, followed by a solution of 412 mg' of 4-hydroxy-3, 5-diisopropylbenzaldehyde in 5 ml of chloroform. The mixture was stirred overnight at room temperature, after which insoluble matter was removed by filtration; the filtrate was then evaporated to dryness at room temperature under reduced pressure,giving 930 mg of lithium 3-acetoxymethyl-7/Ϊ- (4-hydroxy- 3, 5-diisopropylbcnzylideneamino)-3cephem-4-carboxylate, as a yellowish brown powder.

A solution of 4G7 mg of ibis lithium salt in 10 ml of anhydrous tetrahydrofuran was cooled to -40°C-; to this cooled solution were added 3 ml of methanol containing 7 mg of dissol ved metallic lithium, followed by 130 mg of t-butyl hypochlorite. The mixture was stirred, with cooling, for 40 minutes, after which it was evaporated to dryness under reduced pressure, to give the desired lithium 3-aceioxymethyl-7p-(4-hydroxy-3, 5-diisopropylbenzylideneamino)-7a'-methoxy-3-cephcm-4-carboxylate.

NMR spectrum (DMSO-d../CDCl„) 6 ppm: U v (DMSO = dimethyl sulphoxide) 8.48 (singlet, -CIT=N-); 7.55 (singlet, benzene ring II); 5.03 (singlet, ii at 0- position); 5.03 (broad singlet, at 3- position): 43191 3.57 (singlet, OCHg); 3.05 - 3.65 (multiplet, Hg at 2- position. 2,07 (singlet, CHgCO); Q f!TT 1.27 (doublet CH ).

Thin layer chromatography (silica gel) developing solvent chloroform/methanol (3 ; 1 by volume); Rf; 0.44.

PREPARATION 3 Dicyclohexylammonium 7g-(4-hydroxy-3,5-di-t-butylbenzyl· ideneamino)- 7 a- methoxy- 3- (1 -methyl-1 H-tetrazol- 5 -ylthiomethyl)- 3-cephem-4~carboxylate 3.284 g of 70-amino-3-(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-4-oarboxylic acid were suspended in ml of methanol; to the suspension were added 1.81 g of dicyclohexylamine and the mixture was stirred for 1 hour, after which 2.40 g of 4-hydroxy-3,5-di-t-butylbenzaldehyde, 20 ml of chloroform and 5 g of molecular sieve 3A were added to the mixture and stirring was continued for a further hours., 'flic reaction mixture was filtered and the filtrate was evaporated to dryness under reduced pressure,, The residue was extracted with a 1 ; 1 (by volume) mixture of chloroform and isopropyl ether and the extract was condensed under reduced pressure and then diluted with a large excess of isopropyl ether. The precipitate thus produced was collected by filtration, giving t 7, 04 g of dicyclohexylammonium 7j3-(4-liydroxy-3, 5-di-tbutylbenzylideneamino)-3--(1-methyl-lH-tetrazol-5-yl thiomethyl)-3-cephem-4-carboxylate.

A solution of 1.45 g of this compound in 20 ml of methanol was cooled to -15°C, and a solution of 475 mg of 2, 3-dichloro-5, 6-dicyano-l, 4-benzoquinone in 4 ml of methanol was added dropwise. When the addition was complete, the reaction mixture was stirred for 10 minutes at -15°C and there were then added 100 mg of 2, 6-di-tbutyl-p-cresol; stirring was continued for a further 10 minutes at 0°C. At the end of this time, 0. 56 ml of triethylamine was added to the reaction mixture, which was then condensed to about 10 ml by evaporation under reduced pressure. The condensed reaction mixture was diluted with 50 ml of chloroform, washed four times witli the same volume of water, and dried over anhydrous sodium 2191 Sulphate. The solvent was then evaporated off from the mixture under reduced pressure and the residue was extracted with a 3 : 7 (by volume) mixture of chloroform and diethyl ether. The extract was concentrated and a large excess of n-hexane was added to give a precipitate, which was collected by filtration and yielded 926 mg of dicyclohexylammonium 7/3-(4-hydroxy-3, 5-di-t-butylbenzylidene amino)-7ff-methoxy-3-(l-methyl-lH-tetrazol-'5-ylthiomethyl)3-cephem-4-carboxylate, as a yellow powder.

UV spectrum (THK) : 285 nm.

NMR spectrum (CDClg) δ ppm: 8.47 (singlet, -CH=N-); 7.61 (singlet, benzene ring H); .60 (broad, OH); 4.98 (singlet, Hat 6- position); 4. 42 (singlet, -CH^- at 3- position); 3.80 (singlet, N-CHg); 3.48 (singlet, OCHg); around 3.4 (double doublet, Hg at 2- position); 1. 35 (singlet, t-butyl).

Thin layer chromatography (silica gel) developing solvent chloroform/methanol (4 : 1 by volume): R. 0.72.

PREPARATION 4 Diisopropylammonium 7/3-(4-hydroxy-3, 5-di-t-butylbcnzylideneamino)-7a-methoxy-3-(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate 16.5 g of 7/3-amino-3-(l-methyl-in-tetrazol-5-ylthioinethy].)-3-cephem-4-carboxylic acid were suspended in 250 ml of methanol; to the suspension were added 4.545 g of diisopropylamine and the mixture was stirred for 15 minutes. To the mixture were then added 14.1 g of 4-hydroxy-3, 5-dit-butylbenzaldehyde and GO ml of chloroform and stirring was continued for a further 2 hours. At the end of this time, 30 g of anhydrous magnesium sulphate were added to the mixture, which was then stirred for a further 5 hours.

The reaction mixture was then filtered and the filtrate was concentrated by evaporation under reduced pressure to a volume of about 100 ml. 'fhe resulting concentrated Solution was cooled to -15°C, after which 10 g of 2, 3-dichloro-5, C-dicyano-1,4-benzoquinone in 50 ml of methanol were added dropwise; the mixture was then stirred at-15°C for 10 minutes. At the end of this time, 3,1 g of 2, 6-di-t-bulyl-p-cx‘esol were added to the mixture, which was stirred, with ice-cooling, for a further 10 minutes. 11.8 ml of diisopropylamine and 400 ml of benzene were then added to the mixture, which 1 was washed twice with water. The washings were extracted with 100 ml of benzene and the extracts were combined with the washed benzene solution. The combined benzene solution was dried over anhydrous magnesium sulphate and then concentrated to 150 ml. The mixture was poured into 500 ml of n-hexane and the resulting precipitate was collected by filtration to give 19.1 g of the desired diisopropylammonium 7β- (4-hydroxy- 3,5 - di-t-butylbenzylideneamino)-7 UV spectrum (THE) max : 287 nm.

NMR spectrum (CDClg) δ ppm; 8. 54 (singlet. -CH=N-); 7.70 (singlet, benzene ring H); 5.66 (broad, OH); 5.05 (singlet, H at 6- position); 4.50 (singlet, -CHg- at 3- position); 20 3.85 (singlet, N-CHg); 3.54 (singlet, O-CHg)i 3. 63 and 3, 36 (double doublet, Hg at 2-position); 1,43 (singlet, t-butyl). 2191 •PREPARA'IRON 5 Trimothylbenzylammonium 7β-(4-hydroxy-3, 5-di-t-butylbenzylidenc:amino)-7o'-metlioxy-3-(l-methyl-ΙΗ-tctrazol- 5ylthiomcthyl)- 3-copl i cm-4·- carbo xylate 984 mg of 7-amino-3-(l-niethyl-lI!-tetrazol-5-ylthiomcthyl)-3-cephcin-4-carboxylie acid were added to a mixture of 8 nil ol' methanol and 13 ml of letrahydrofuran, with stirring and ice-cooling; 1.36 ml of a 40 % melhanolic solution of TRITON II (a trade mark for trimethylbe'nzylammonium hydroxide) were added to the resulting mixture. The mixture was allowed to reach room temperature, at which it became a homogeneous solution; 712 mg of 4-hydroxy-3, 5-dj-t- mitylberizaldehyde and 2 g of anhydrous calcium sulphate we ό added to this solution and the resulting mixture was stirred overnight at room temperature. After fill ration of the reaction mixture, the solvent was evaporated from the filtrate, under reduced pressure, giving trimethylbenzy'ammonium 7/?-(4-hydroxy-3, 5-di-t-butylbenzylidenecmino)- 3-(1-methyl 1 Π-tetrazol-5 -ylthio methyl) - 3- cephem-4- carboxylate in quantitative yield as a pale brown powder.

A solution of 2.17 g of this trimcthylbenzylonimonium 7/3-(4-hydroxy- 3, 5-di-t-butylbenzylideneaniino)-8-(1-methyl 31 in-tetrazol-5-ylthioinethyl)-3-ceplKim-4-carboxy)atc in 13 ml of methanol and 13 ml of tetrahydrofuran was stirred and cooled in a bath of dry ice and methanol; to the cooled solution were added 4 ml of methanol containing 42 mg of dissolved metallic lithium, after which a solution of 780 mg of t-butyl hypochlorite in 2 ml of diehloroethane was added dropwise to the mixture. Stirring was continued for 35 minutes, whilst cooling the reaction mixture, and then the mixture was condensed to about 5 ml by evaporation at room temperature under reduced pressure, ml of chloroform were added to the condensed mixture.

The mixture was then washed four times with an aqueous sodium chloride' solution, dried over anhydrous magnesium , sulphate and then condensed to about 5 ml. Cyclo15 hexane was added to the residue, which was then evaporated to dryness, giving 1.5 g of trimethylbenzylammonium 7β(4-hydroxy-3, 5-di-t-butylbenzylideneamino)-3-(l-methyllH-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate, as a pale yellow powder.

Thin layer chromatography (silica gel) Developing solvent : chloroform/methanol (3 : 1 by volume) Rf = 0,53. 42l9i EXAMPLE 1 methoxy-3-ccphem-4-carboxyIic acid (Compound 3) 520 mg of lithium 3-acetoxymethyl-7/?-(4-hydi-oxy5 3,5-diisopropy]bcnzylideneaniiiio)-7o'-melhoxy-3-ccphem4-carbc>xylate, prepared by the method described in Preparation 2, were dissolved in 4 ml of methanol, and 185 mg of Girard T reagent were added whilst Stirring the mixture and cooling it in an ice/salt bath. After further stirring the mixture for 10 minutes, with cooling, solutions of 450 mg of N, N-diethylaiiiline in 1 ml of methanol and 320 mg of cyanomethylthioacetyl chloride in 1 ml of dichloroethane were successively added dropwise. Stirring was continued for 30 minutes whilst cooling the reaction mixture in the ice/salt bath. 20 ml of 0.2N hydrochloric acid were then added to the reaction mixture thus obtained, the mixture was stirred for 2-3 minutes and it was then extracted with ethyl acetate.

The extract was washed with water and the acidic substance in the extract was separated by slinking three times with a % aqueous solution of dipotassium hydrogen phosphate.

The aqueous phase was washed twice with ethyl acetate and its pll was then adjusted to a value of 2. 0 by adding 10 % hydrochloric acid. The resulting solution was again extracted with ethyl acetate, the extract was dried over anhydrous magnesium sulphate, and the solvent was removed by evaporation under reduced pressure.

The residue was washed with oyclohexane and a small amount of diethyl ether, yielding 95 mg of 3-acetoxymethyl7)3- cyanomethylthioacetamido- 7 a- methoxy- 3- cephem-4carboxylic acid (Compound 3).

IR spectrum (Nujol'- trade mark) > 3280, 2250, 1775, 1735, 1690.

UV spectrum (buffer solution, pH 6.8)^, nm: 247, 267. max NMR spectrum (CDgCN) δ ppm: ,06 (1H, singlet, H at 6- position); 4.79 - 5.06 (2H, quartet, -CH^- at 3- position); 3.60 (2H, singlet, -CHgSCHgCO- or -CHgSCHgCO15 at 7- position); 3.52 (3H, singlet, 0CHg at 7-position); 3.47 (2H, singlet, -CHgSCHgCO- or -CHgSCHgCO- at 7- position); 3. 32 - 3. 55 (2H, quartet, H^ at 2- position); 2,02 (3H, singlet, -OCOCIIg at 3- position).

EXAMPLE 2 7/l~Cyanometliylthioacetamido-7c-methoxy-3-(1-methyl 111-tetrazol-5-ylthiomethyl)-3-eephem-4-carboxylic acid (Compound 16 ) .45 g of dicyclohexylammonium 7β-(4-hydroxy-3, 5di-t-butylbenzj'lideneamino)-7t?-methoxy-3-(l-methyl-lIItetrazol-5-ylthiomethyl)-3-copbem-4-carboxylatc, prepared as described in Preparation 3, were dissolved in 150 ml of methylene chloride, and 8. 38 g of phenylacotic acid hydrazide were added to the resulting solution; the mixture was then stirred for 75 minutes at room temperature.

At the end of this time, the mixture was cooled to -15°C, 22,4 ml of di ethyl aniline and 21.0 g of cynnomethylthioacctyl chloride were added, and the mixture was stirred for minutes vzith ice-cooling. The reaction mixture was then concentrated by evaporation under reduced pressure and diluted with ethyl acetate, which caused a crystalline substance to deposit; this crystalline substance was collected by filtration. The remaining ethyl acetate solution was washed with a small quantity of water and then extracted twice with a 10 % aqueous solution of dipotassium hydrogen phosphate. Ethyl acetate was added to the aqueous phase and the pi I of the mixture was adjusted to a value of 2.5 by addition of 3N hydrochloric acid, after which the mixture was shaken well. The aqueous phase which separated out was, after saturation with sodium chloride, repeatedly extracted with ethyl acetate. The combined extracts were washed twice with a saturated aqueous solution of sodium chloride and once with a small 5 quantity of water; the washed extracts were then dried over anhydrous sodium sulphate. · Removal of the solvent by evaporation under reduced pressure yielded 16. 04 g of Compound 16' as a syrupy substance.

NMR spectrum (CDgCN+DgO) δ ppm: .10 (singlet, H at 6-position); 4. 3 - 4,6 (quartet, -CIL- at 3- position); 3, 98 (singlet, Ν-ΟΗθ at 3- position in tetrazole), 3.70 (singlet, -CHgSCIIgCO- or -CIlgSCHgCO- at 7- position); 3.60 (singlet, -CH.SCH.CO- or -CHgSCHgCO- at 7- position); 3. 5 - 3. 7 (multiplet, Hg at 2- position); 3.50 (singlet, OCHg at 7-position).

EXAMPLE 3 Sodium 7/3-cyanoroethylthioacetan)ido -7a1-methoxy- 3-(1 -methyllH-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate To 500 mg of 7/3-cyanomethylthioacetamido-7n36 43191 methoxy-3-(1 -inelhyl-1 H-tolrazol-5-ylthiomctliyl)-3cephom-4-carboxylic acid, prepared as described in Example 2, in 4 ml of acetone was added 1 ml of a butanol solution containing 1.1 times the equimolar amount of sodium 2-ethylhexanoate. To the solution were added 7 ml of diethyl ether and the precipitate so produced was filtered off and washed witli diethyl ether, giving 500 ml of the desired salt.

NMR spectrum (DgO) δ ppm: , 20 (singlet, II at G- position); 4. 22 (quartet, -dig nt 3- position); 4.06 (singlet, N-CIIg); 3.73 (singlet, -CH2SCII2CO- at 7-position); 3.68 (singlet, -CllgSCII^CO- at 7-position); 3.60 (singlet, -OC'lIg at 7- position); 3.55 (quartet, 11^ nt 2- position).

EXAMPLE 4 Dicyclohexy] amine 7/)-cyaiiometli.ylthioac tamido - To-methoxy3-(l-niethyJ-in-tetrnzol-5-ylthiomothyI) 3-cephem-4-earboxylate To 90 mg of 7/3-cyanomethylthioai etnmido- 7a-methoxy~ 3-(1-methyl-in-tetrazol-5-ylthiomcthyl)· 3-cephem-437 i carboxylic acid, prepared as described in Example 2, in 0.5 ml of acetone was added 0. 3 ml of an acetone solution containing 40 mg of dicyclohexylamine.

To this solution were then added 5 ml of diethyl ether and the precipitate produced was filtered off and recrystallized from ethanol, giving 70 mg of the desired salt melting at 155°C (with decomposition).

EXAMPLE 5 3-Acetoxymethyl-73-cyanomethylthioacetamido - 7a-methoxy10 3-cephem-4-carboxylic acid (Compound 3) To a solution, of 14.0 g of dicyclohexylammonium 3-acetoxymethyI-7)3-(4-hydroxy-3, 5-di-t-btetylbenzylideneamino)7 a-methoxy-3-cephem-4-carboxylate (prepared as described in Preparation 1) in 70 ml of methylene chloride were added 3,60 g of phenylacetic acid hydrazide, and the mixture was stirred for-70 mihutes at room temperature. The reaction mixture was then cooled to-15°C, after which 9,51 ml of -.diethylaniline and 9.0 g of cyanemethylthioaeetyl chloride were added and the mixture was stirred for 40 minutes, with ice-cooling. 50 ml of methanol were then added to the reaction mixture, which was stirred for a further 2 hours at room temperature, after which it was concentrated by evaporation wider reduced pressure and diluted with ethyl • 38 acetate to deposit a crystalline substance, which was removed by filtration.

The ethyl acetate solution was washed with a small quantity of water and then extracted three times with a 10 % aqueous solution of sodium phosphate. The pH of the combined extracts was adjusted to 2.5 by addition of 3N hydrochloric acid and the oily substance which separated was then extracted with ethyl acetate. The aqueous phase was also thoroughly extracted with ethyl acetate. The combined ethyl acetate extracts were washed with water and the washings were extracted with ethyl acetate. The ethyl acetate solutions were dried over anhydrous sodium sulphate and the solvent was then removed by distillation under reduced pressure, giving G.53 g of Compound 3 as a syrupy substance. The properties were as described in Example 1.

EXAMPLE 6 3-Acetoxymethyl-7)3-isoxazol-3-ylthioacetamido-7ff-methoxy3-ccpliem-4 -carboxylic acid (Compound 7) The procedure described in Example 5 was repeated, except that the cyanomethylthioacetyl chloride was replaced by 10,9 g of isoxazol-3-ylthioacetyl chloride, giving 6.70 g of Compound 7, melting point 130 - 132°C (with decomposition).

IR spectrum (Nujol - trade mark) cm : 3500, 3270, 1780, 1730, 1700.

UV spectrum (buffer solution, pH 6.8) max nm: 261 ( £ = 8000).

NMR spectrum (DMSO-άθ) δ ppm: 9.34 (singlet, -CONH- at 7- position); 8. 63 (doublet, H at 5- position of isoxazole); 6. 34 (doublet, H at 4- position of isoxazole); .15 (singlet, H at 6-position); .12-4.64 (couple of doublet, -CH^-O- at 3-position); 5,01 (singlet, -S-CHgCO- ); 3.68 -3.14 (couple of doublet, Hg at 2- position); 3,43 (singlet, -OClIg at 7- position); 2. 02 (singlet, -COCIIg at 3-position).

EXAMPLE 7 3-Acetoxymethyl-7j3-azidomethylthioacetamido-7Q,-methoxy3-cephem-4-carboxylic acid (Compound 1) The procedure described in Example 5 was repeated, 4 21 9 I except that the cyanomethylthioaceVyl chloride was replaced by S). 93 g of azidoinclhylthioacetyl chloride, giving 7. 37 g of 3-acetoxymethyl-7/l-azidoniethylthioacetamido-7a-methoxy3-cephem-4-carboxylic acid (Compound 1). ΙΠ spectrum (Nujol - trade mark) cm : max 3280, 2110, 1775, 1730, 1720(sh), 1690. (sh = shoulder). UV spectrum(C2H5OII) Kax® 247 (£. = 7,800), 269 (E = 8,000). 10 NMll spectrum (DMSO-dg) δ ppm: 5.14 (in, singlet, II at 6- position); 4.C9- 4. 98 (211, quartet, CH^OCO- at 3- position); 4. 51 (211, singlet, NgCIIgS-); 3.40 (5H, singlet, -OCTI at 7-position and SCH„CO), u T 15 3.3 - 3.6 (2Π, quartet, H„ at 2- position); u 2. 00 (311, Kinglet, -OCOCIIg).

Elemental ansilysis; Calculated for C NgO? Sg : C, 38.97 %; II, 3.97%; N, 16.23%, 20 Found: C, 39. 08 %; H, 4.21%; N, 15,88%.

EXAMPLE 8 3-Acetoxymethyl-7P-isoxazol- 3-yloxyacetamido-7g-methoxy 3-cephem44-carboxylic acid (Compound 6) The procedure described in Example 5 was repeated, S except that the cyanomethylthioacetyl chloride, was replaced by 9, 7 g of isoxazol-3-yloxyacetyl chloride, giving 6, 57 g of 3-acetoxymethyl-7/3-isoxazol~3-yloxyacetamido~7a-methoxy3-cephem-4-carboxylic acid.

IR spectrum (Nujol - trade mark) maxcm”^: 3500, 3270, 1780, 1725, 1700 (sh). UV spectrum (buffer solution, pll 6.8) X nm: 264 (£ = 8, 300). NMR spectrum (DMSO-dg) 5 ppm: 9. 56 (1H, singlet, -CONTI- at 7- position); 15 8.70 (1H, doublet H at 5- position of isoxazole); 6. 31 (1H, doublet, H at 4- position of isoxazole); 5.30 (1H, singlet, II at 6- position); 5,12 - 4,18 (2H, double doublet, -CII^O at 3-position). 4.82 (2H, singlet, -OCI^CO); 20 3.85 - 3.14 (2H, double doublet, -CHg- at 2- position); 3.41 (3H, singlet, -OCHg at 7- position); 2.00 (311, singlet, -COGIIg at 3- position).

Thin layer chromatography (silica gel)· Developing solvent: methanol/chloroform (1 : 2 by volume): Rf = 0,28, 4219 1 Elemental analysis Calculated for Cg Jig Og Ng S: C, 44.97 %; Π, 4.03 %; N, 9.83%. Jibund: C, 45.12%; II, 4.36%; N, 9.06%.

EXAMPLE 9 3-Acct0xymcthyl-7o,-mcthoxy-7(I-propargylthioacetamido3-cephem'-4-<:arboxylic acid (Compound 6) The procedure described in Example 5 was repeated, except that the cyanornethyllhioat·elyl chloride was replaced by 6.85 g of propargylUiioacetyl chloride, giving 6.50 g of 3-acetoxymethyl-7c-methoxy~7/l-propargylthioacetamido-3cephem-4-carboxylic acid (Compound 8), having a melting point of 122 - 124°C (with decomposition). lit spectrum (KBr) JS cn? : 1 max 3260, 1775, 1730, 1705, 1660.

UV spectrum (C-1Γ.ΟΙΙ) r nm: 245 (¢= 7,800), 268 (6--= 8, 200).

NMR spectrum (I)MSO-d ) 6 ppm: ,05 (111, singlet, 11 at 6- position); 4. 9 -4.6 (2Π, quartet, -ClI OCO- at 3-position); 3,36 (311, singlet, -OCHg at 7-position); 3.2-3.5 (611, multiplet, Hg at 2- position and -CH2SCH2-); 3.05 (1H, triplet, HC s C- ); 1.98 (3H, singlet, OCOCHg ).

Elemental analysis Calculated for C, a H, _ O„ S„ : 18 7 2 2 C, 46.36 %; H, 4.38%; N, 6.76%.

Found: C, 46.21 %; H, 4.30 %; N, 6.55%, EXAMPLE 10 7/J-Cyanomethylthioacetamido-7g-methoxy-3-(l-methyl-lHtetrazol-5-ylthiomethyl)- 3-cephem-4-oarboxylic acid (Compound 16) (a) Trimethylbenzylammonium7P-amino-7g-methoxy3-(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem15 4-carboxylate 1,5 g of trimethylbenzylammonium 7|3-(4-hydroxy3,5-di-t-butylbenzylideneamino)-7g-methoxy-3-(1-methyllH-tetrazol-5-yltliiomethyl)-3-cephem-4-carboxylate, prepared as described in Preparation 5, were dissolved in ml of dichloroethane, and 0,8 g of phenylhydrazine in ml of dichloroethane was added, whilst stirring and maintaining the temperature at 0 - 5°C. After 4219 1 minutps, 40 nJ of cyclohexane were added to the reaction mixture and the precipitate which was produced was collected I by filtration and washed witli a mixture of cyclohexane and diethyl ether (1 : 1 by volume) to give 1.03 g of trimethylbenzylaninioiiium 7^-amino-7a-methoxy-3-(l-methyl-intetras5ol-5-ylthiomethyl)-3-cephem-4-carboxylate.

Thin layer chromatography (silica gel) Developing solvent: chloroform/methanol (3 : 1 by volume): Rf = 0.1.

This ammonium salt was used jier mi for the next step, but the purified free carboxylic acid may be obtained by chromatography using dried silica gel eluted with a 10 : 1 by volume mixture of chloroform and methanol and’Bephadcx LU-20 eluted witli a 10 : 1 by volume mixture of chloroform and methanol; 0. 2 g of the purified free carboxylic acid were obtained.

IR spectrum (Nujol - trade mark) J/ cm : ΙΪ13Χ 3300-3400 (NH2), 1740-1760 (β lactam).

NMR spectrum (DMSO-d^) δ ppm; 4.64 (ID, singlet, II at 6- position); 4.15 (211, broad singlet, -CII^S- at 3- poi 3.80 (3Π, singlet, N-CHg); 3.40 (2IT, quartet, 11 at 2- position); 3, 22 (3Π, singlet, OCI(g at 7- position), *Sephadex is a trade mark f 43151 (b) 7/l-CyanomethyIthioacetamitlo-7ft-nietl)oxy-3-(l -methyllII-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylxc acid The 1, 03 g of trimethylbenzyl ammonium 7j3-amino7a-methoxy-3-(1-methyl-ΙΗ-tetrazol-5-ylthiomethyl)-35 cephem-4-carboxylate prepared as described above were dissolved in 14 ml of dichloroethane; to the resulting solution was added a solution of 0. 9 g of N, N-diethylaniline in 2 ml of diehloroethane, followed by a solution of 0. 9 g of cyanomethylthioacetyl chloride in 2 ml of diehloroethane, with stirring and cooling with a freezing mixture.

After stirring the reaction mixture for 40 minutes, 20 ml of methanol were added and stirring was continued for a further 1 hour. The reaction mixture was then concentrated by evaporation at room temperature, and the residue was dissolved in 10 ml of chloroform and 30 ml of a 10 % aqueous solution of dipotassium hydrogen phosphate and stirred for 10 minutes. The chloroform layer which separated out was extracted twice with a 10 % aqueous solution of dipotassium hydrogen phosphate, The combined aqueous extracts were washed twice with ethyl acetate and the pH was adjusted to a value of 2, 0 by addition by 3N hydrochloric acid. An oily substance separated out and was extracted with ethyl acetate; the extract was washed once with water and dried over anhydrous magnesium sulphate, after wiiich the solvent was removed to give 70G mg of 7(3-cyanomethyltbioacetamido-7a-methoxy 3-(1-methyl-l I_I-tetrazol-5-yliliiometliyl)-3-cephem-4carboxylic acid, in crude form, as a pale brown powder.

A solution of this crude product in 4 ml of ethyl acetate was cooled with ice and then 0.45 ml of dicyclohexylamine was added,forming a crystalline product, which, after addition of diethyl ether,was collected by filtration and recrystallized from ethanol to give 473 mg of the dicyclohexylamine salt, as colourless crystals melting at 152°C (with decomposition). To this crystalline salt were added about 10 ml of water and 20 ml of ethyl acetate and the pll of the mixture was adjusted to a value of 2 - 1.5 by addition of 10 % hydrochloric acid, with stirring. The mixture was shaken in a separating funnel and the ethyl acetate layer was separated off. The aqueous layer was extracted three times with ethyl acetate and the ethyl acetate extracts were combined with the original ethyl acetate layer and the whole was washed once with water and dried over anhydrous magnesium sulphate; the solvent was then removed, giving 240 mg of 7/l-eyanomethylthioacetamido-7a-methoxy-3-(lmethyl-lH-teirazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid as a nearly white powder.

EXAMPLE 11 7o'-Methoxy-3*{l-methyl-in-tetraKol-5-yTthioitiel:hyl)-7/3propargylthioacetamido-3-cephem-4-carboxylie acid (Compound 23) 6. 76 g of diisopropylammonium 7/3-(4-hydroxy-3, 5di-t-butylbenzylideneamino)-7a-methoxy-3-(1-methyl-lHtetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate (prepared by the method described in Preparation 4) were dissolved in 25 ml Of methylene chloride; to the resulting solution was added 1.08 g of phenylhydrazine and the mixture was then stirred, with ice-cooling, for 20 minutes. At the end of this time, the reaction mixture was cooled to -15°C and 4.80 ml of diethylaniline and 4.45 g of propargylthioacetyl chloride were added to the mixture. The mixture was then stirred, with ice-cooling, for 40 minutes, after which 30 ml of methanol were added and the mixture was stirred for a further 2 hours at room temperature.

The mixture was then evaporated to dryness under reduced pressure and the residue was dissolved in ethyl acetate.

The resulting solution was washed with a small quantity of water and extracted three times with a 10 % aqueous solution of disodium hydrogen phosphate. The extracts were combined and their pH adjusted to a value of 2.5 by addition of 3N hydrochloric acid. The oily substance which was thereby produced was extracted with ethyl acetate. 421y i The extract was washed with water and dried over anhydrous sodium sulphate; the solvent was then distilled off, giving 2.72 g of Compound 23, as a syrupy substance.

NMR. spectrum (CD CN+D Ο) ΰ ppm: ,10 (singlet, II at 6-position); 4.3-4,6 (quartet,-CH- at 3-position); 3.98 (singlet, N-CH at 3- position in tetrazole); 3.70 (singlet, -C'H SCII CO- or-CIIgSCHgCOat 7- position); 3.60 (singlet, -CH.SCH CO- or -CH„SCH„CO—“Δ Δ T <5 at 7- position); 3.5-3.7 (multiplet, Ilg at 2-position); 3.50 (singlet, -OCHg at 7-position).

Following the procedure described in Example 11, the following compounds were obtained by using the following substituted acetyl chlorides in place of propargylthioacetyl chloride: 2, 67 g of 7a-methoxy-3-(l-methyl-lH-tetrazol-5ylt hiomethyl) - 7/3 - sy dn on - 3 - ylac e t a mi d o - 3 - c eph era - 4 carboxylate (Compound 24) were obtained from 4.89 g of sydnon-3-ylacetyl chloride. 2. 91 g of 7/3-(l-cyarioethyllhioaeet;imido)-7Q'~mcthoxy3-(1 -methyl - ΙΗ-tetrazol- 5-ylthion lethyl)- 3-cephem-4 49 43191 carboxylic acid (Compound 15) were obtained from 4. 90 g of 1-cyanoethylthioacelyl chloride, 2. 92 g of 7a-methoxy-7/?-methylsulphonylacetamido3-(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-45 carboxylic acid (Compound 22) were obtained from 4. 69 g of methylsulphonylacetyl chloride. 2.85 g of 7/?-ethylsulphonylacetamido-7a-methoxy3- (l-methyl-lH-tetrazol-5ylthiomethyl)“3-cephem-4carboxylic acid (Compound 17) were obtained from 5.11 g of ethylsulphonylacetyl chloride. 3.05 g of 73-cyanoethylsulphonylacetamido-7nmethoxy-3t-(l-methyl-lH-tetrazol-5-ylthiomethyl)~3-cephem4- carboxylic acid (Compound 14) were obtained from 5. 86 g of cyanoethylsulphonylacetyl chloride. 2.53 gof7B-azidomethylthioacetamido-7oi-methoxy3-'(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-4carboxylic acid (Compound 13)wereprepared from 3.70 g of azidomethylthioacetyl chloride. 2.60 g of 7/3-isoxazol-3~yloxyacetamido-7ff-methoxy20 3-(l-methyl-in-tetrazol-5-ylthiomethyl)-3~eephem~4carboxylic acid (Compound 20) were prepared from 4.85 g of isoxazol- 3-yloxyacetyl chloride. 2.91 gof7/3-isoxazol-3-ylthioacetamido-7a-methoxy3-(1 -methyl- lH-tetrazol-5-yltbi omethyl)-3-cephem~4carboxylic acid (Compound 21) were prepared from 5.41 g of isoxazol-3-ylthioacetyl chloride.

EXAMPLE 12 7/3- (2-Hydroxyethyl)thioacetamido-7 a- methoxy- 3-(1 -methyl lH-tetyazol-5-ylthiomethyl)- 3-cephem-4-carboxylic acid (Compound 18) The procedure described in Example ll was repeated, except that the propargylthioaeetyl chloride was replaced by 7.18 g of tetrahydropyranyl-2-oxyethylthioacetyl chloride; there were obtained 2.52 g of 7a-methoxy-3-(l-methyl-lHtetrazol-5-ylthiomethyl) - 7/3-(tetrahydropyranyl-2 - oxyethyl15 thioaeetamido)-3-eephem-4-earboxylic acid, as a pale yellow powder. This powder was dissolved in 10. ml of anisole, and 10 ml of trifluoroacetic acid were added to the solution, with ice-cooling, after which the mixture was stirred at room temperature for 5 minutes, The solvent was then distilled off and the residue was dissolved in a mixture of 200 ml of ethyl acetate and 50 ml of a 10 % aqueous solution of dipotassium hydrogen phosphate.

The aqueous layer was washed with 10 ml of ethyl acetate, its pH was adjusted to a value of 2. 0 , it was saturated with sodium chloride and then extracted with ethyl acetate.

The extract was dried and the solvent was distilled off.

The residue was purified by preparative thin layer chromatography, giving 670 mg of Compound 18 as an amorphous powder.

IR spectrum (KBr) cm~^ : max 1765, 1675.

UV spectrum (phosphate buffer, pH· 6.86 ) maxnm270 ( £= 9450 ).

Claims (20)

1. CLAMS

1. A process for preparing a 7j3-amino-7ff-methoxy3-cephem-4-carboxylate of general formula (III): 5 (in which: R* represents an acetoxy group, a carbamoyloxy group or a 1-methyl-111-tetrazol-5-ylthio group; and Y represents a cation), which comprises reacting a 7/3-arylmethyleneamino-7(v-rnethoxy-3-cephem-4-carboxylate of general formula (II): (in which R^ and Y + are as tiefined .ibove; n is 1 or 2; and Ar represents a substituted or unsubstituted, monocyclic or fused polycyclic benzenoid ring system) with a hydrazine compound.

2. A process according to Claim 1, in which Ar represents the group: 2 3 4 5 in which R , R , R and R are the same or different and each represents a hydrogen atom, an alkyl group having from 1 to 4 carbon atoms, an alkoxy group having from 1 to 4 carbon atoms, a halogen atom, a cyano group or an 10 alkoxycarbonyl group having from 1 to 4 carbon atoms 2 3 4 in the alkoxy moiety, or R and R together and/or R g and R together form, together with the carbon atoms to which they are joined, a carbocyclic or heterocyclic, saturated, unsaturated or aromatic ring. 2 5 15 3. A process according to Claim 2, in which R and R

3. 4 each represents a hydrogen atom, R and R each represents an isopropyl or t-butyl group and n is 1. 421S1

4. A process according to any one of Claims 1 to 3, in which Y + represents an alkali meta] ion. +

5. A process according to Claim 4, in which Y represents a sodium or lithium ion. 5

6. A process according to any one of Claims 1 to 3, + in which Y represents an ammonium ion having the formula: f b RC-N + -Ra in which R a , r\ R c and R^ are the same or different 0 and each represents a hydrogen atom, a straight or branched chain alkyl group having from 1 to 8 carbon atoms, a cycloalkyl group having from 5 to 7 carbon atoms, a phenyl group or a phenylalkyl group having from 1 to 4 carbon & b a atoms in the alkyl moiety, or R and R together or R , r b c J R and R together form, together with the nitrogen atom to which they are joined, a heterocyclic ring.

7. A process according to Claim 6, in which Y + represents a t-butylammonium ion, a t-octylammonium ion, a dicyclohexylammonium ion, a diisopropylammonium ion, a triethylammoniuin ion or a trimethylbenzylammonium ion.

8. A process according to any one of Claims 1 to 7, in which said hydrazine compound is.phenylaeetic acid hydrazide, hydrazine, hydrazine hydrate, phenylhydrazine, 5 2,4-dinitrophenylhydrazine, carboxymethylpyridinium chloride hydrazide or carboxymethyltriraethylammonium chloride hydrazide.

9. A 7/3-amino-7a-methoxy-3-cephem-4-earboxylate of general formula (III) when prepared'by a process 1° according to any one of Claims 1 to 8.

10. . A process for preparing a 7ff-methoxyeephalosporin of general formula (I): Π) (in which: R is as defined in Claim 1; and R represents a cyanomethylthio group, a l-cj'anoethylthio group, an 4 2191 azidomethylthio group, a propargylthio group, a 2-hydroxyethylthio group, a 2, 3-dihydroxypropylthio group,a inethylsulphonyl group, an ethylsulphonyl group, a cyanoethylsulphonyl group, an isoxazol-3-yloxy group, an isoxazol-3-ylthio group, 5 a 1, 3,4-thiadiazol-2-ylthio group, an imidazol-2-ylthio group or a sydnon-3-yl group) which comprises reacting a 7/3-amino7o-methoxy-3-cephem-4-carboxylate according to Claim 9 with a carboxylic acid of general formula (IV): R 6 CII COOII (IV) (in which lA represents the group It or the group It in which any hydroxy groups have been protected) or with a functional equivalent of said acid (IV) to produce a compound of general formula (V): r g cii 2 coiihoch 3 c i COOH ΓΗ 2 β Ί (V) 1 6 (in which It and It are as defined above) and, if necessary, removing any protecting groups in the group 1{θ.

11. A process according to Claim 10, in which said 4219 t functional equivalent of said carboxylic acid (IV) is an acid halide, an acid azide, a mixed acid anhydride, an acid anhydride or a reactive ester.

12. A process according to Claim 11, in which said 5 functional equivalent is an acid halide and the reaction is effected in the presence of an acid binding agent.

13. A process according to Claim 12, in which said acid binding agent is an organic or inorganic base.

14. A process according to Claim 13, in which said 10 '' organic base is a tertiary amine.

15. A process according to Claim 14, in which said tertiary amine is diethylaniline,

16. A process according to any one of Claims 11 to 15, ir. which said acid halide is an acid chloride, 15

17. A process according to Claim 10, substantially as hereinbefore described with reference to any one of foregoing Examples 1, 2 and 5 to 12. 4219^

18. A 7a-ineliioxycephalosporin compound of general formula (I) when prepared by a process according to any one of Claims 10 to 17. t

19. A process for preparing a salt of a 7ο-melhoxycephalosporin compound according to Claim 18, in which said compound (I) or said compound (V) is salified,

20. , A process according to Claim 1 9, substantially as hereinbefore described with reference to either of foregoing